Methods of antagonizing signal transduction in dorsal root ganglion cells

ABSTRACT

Use of antagonists to IL-31Ra and OSMRb are used to treat inflammation and pain by inhibiting, preventing, reducing, minimizing, limiting or minimizing stimulation in neuronal tissues. Such antagonists include soluble receptors, antibodies and fragments, derivative, or variants thereof. Symptoms such as pain, tingle, sensitization, tickle associated with neuropathies are ameliorated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/332,758, filed Dec. 21, 2011, which is a divisional of U.S.application Ser. No. 13/075,845, filed Mar. 30, 2011, now U.S. Pat. No.8,105,591, which is a divisional of U.S. application Ser. No.12/607,831, filed Oct. 28, 2009, now U.S. Pat. No. 7,943,132, which is adivisional of U.S. application Ser. No. 11/621,840, filed Jan. 10, 2007,now U.S. Pat. No. 7,638,126, which claims the benefit of U.S.Provisional Application Ser. No. 60/758,066, filed Jan. 10, 2006, U.S.Provisional Application Ser. No. 60/757,979, filed Jan. 10, 2006, U.S.Provisional Application Ser. No. 60/773,031, filed Feb. 14, 2006, U.S.Provisional Application Ser. No. 60/805,552, filed Jun. 22, 2006, U.S.Provisional Application Ser. No. 60/805,550, filed Jun. 22, 2006, U.S.Provisional Application Ser. No. 60/805,554, filed Jun. 22, 2006, U.S.Provisional Application Ser. No. 60/823,982, filed Aug. 30, 2006, andU.S. Provisional Application Ser. No. 60/823,987, filed Aug. 30, 2006,all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

The inflammatory process activates the nervous system causinginflammatory pain and a disruption in motor function. Stimulation ofsensory nerves produces vasodialtion and plasma extravasation, leadingto neurogenic inflammation and stimulation causing sensory irritation,hypersensitivity and pain.

Neurogenic inflammation is caused by activation of nociceptive andthermal-sensitive endings in tissues and can be caused by innateconditions, such as autoimmune diseases, including allergy, by viralinfection, as well as by injury. The neurogenic inflammation from theseconditions can affect the somatosensory system, which consists ofvarious sensory receptors responsible for sensations such as pressure,touch, temperature, pain, itch, tickle, tingle, and numbness. Activatednerves can perpetuate chronic inflammation by inducing secretion ofcytokines, activating monocytes and chemotaxis.

Proteins active in neurogenic inflammation can serve as targets fortherapeutic approaches to diagnosis and treatment of diseases.

An example of a drug used to treat pain is Neurontin (gabapentin), whichis used to treat diabetic peripheral neuropathy as post-herpaticneuralgia. Thus, there is a need for additional medication to treatneuropathic pain.

DESCRIPTION OF THE INVENTION

The following definitions are provided to facilitate understanding ofthe inventions described herein.

The term “antibody” or “antibody peptide(s)” refers to an intactantibody, or a binding fragment thereof that competes with the intactantibody for specific binding and includes chimeric, humanized, fullyhuman, and bispecific antibodies. In certain embodiments, bindingfragments are produced by recombinant DNA techniques. In additionalembodiments, binding fragments are produced by enzymatic or chemicalcleavage of intact antibodies. Binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies.

The term “isolated antibody” refers to an antibody that has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In embodiments, the antibodywill be purified (1) to greater than 95% by weight of antibody asdetermined by the Lowry method, and including more than 99% by weight,(2) to a degree sufficient to obtain at least 15 residues of N-terminalor internal amino acid sequence by use of a spinning cup sequenator, or(3) to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

A “variant” anti- IL-31 antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” anti- IL-31 antibodyamino acid sequence by virtue of addition, deletion and/or substitutionof one or more amino acid residue(s) in the parent antibody sequence. Inan embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more hypervariable region(s) of the parentantibody. For example, the variant may comprise at least one, e.g. fromabout one to about ten, and from about two to about five, substitutionsin one or more hypervariable regions of the parent antibody. Ordinarily,the variant will have an amino acid sequence having at least 75% aminoacid sequence identity with the parent antibody heavy or light chainvariable domain sequences, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, and most preferably at least95%. Identity or homology with respect to this sequence is definedherein as the percentage of amino acid residues in the candidatesequence that are identical with the parent antibody residues, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. None of N-terminal, C-terminal,or internal extensions, deletions, or insertions into the antibodysequence shall be construed as affecting sequence identity or homology.The variant retains the ability to bind human IL-31 and preferably hasproperties which are superior to those of the parent antibody. Forexample, the variant may have a stronger binding affinity, enhancedability to inhibit IL-31-induced stimulation of immune cells. To analyzesuch properties, one should compare a Fab form of the variant to a Fabform of the parent antibody or a full length form of the variant to afull length form of the parent antibody, for example, since it has beenfound that the format of the anti-IL-31 antibody impacts its activity inthe biological activity assays disclosed herein. The variant antibody ofparticular interest herein is one which displays at least about 10 fold,preferably at least about 20 fold, and most preferably at least about 50fold, enhancement in biological activity when compared to the parentantibody.

The term “parent antibody” as used herein refers to an antibody which isencoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or human antibody.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule. IL-31 agonists cause, forexample: stimulation of NK cells, T cell subsets and B cell subsets anddendritic cells.

[12] The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule. IL-31Ra and OSMRbantagonists cause: decreased immune function of NK cells, T cell subsetsand B cell subsets and dendritic cells; bind. IL-31 such that theinteraction of IL-31 protein is blocked, inhibited, reduced, antagonizedor neutralized.

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab' fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992).

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. More specifically, the term “IL-31Ra epitope” as usedherein refers to a portion of a IL-31Ra polypeptide having antigenic orimmunogenic activity in an animal, preferably in a mammal, and mostpreferably in a mouse or a human. An epitope having immunogenic activityis a portion of a IL-31Ra polypeptide that elicits an antibody responsein an animal. An epitope having antigenic activity is a portion of aIL-31Ra polypeptide to which an antibody immunospecifically binds asdetermined by any method well known in the art, for example, byimmunoassays. Antigenic epitopes need not necessarily be immunogenic.

The term “epitope tagged” when used herein refers to the anti-IL-31Raantibody fused to an “epitope tag”. The epitope tag polypeptide hasenough residues to provide an epitope against which an antibody can bemade, yet is short enough such that it does not interfere with activityof the IL-31Ra antibody. The epitope tag preferably is sufficientlyunique so that the antibody does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least 6amino acid residues and usually between about 8-50 amino acid residues(preferably between about 9-30 residues). Examples include the flu HAtag polypeptide and its antibody 12CA5 (Field et al. Mol. Cell. Biol.8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto (Evan et al., Mol. Cell. Biol.5(12):3610-3616(1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering3(6):547-553(1990)). In certain embodiments, the epitope tag is a“salvage receptor binding epitope”. As used herein, the term “salvagereceptor binding epitope” refers to an epitope of the Fc region of anIgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsible forincreasing the in vivo serum half-life of the IgG molecule.

The term “fragment” as used herein refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues of the amino acid sequence of a IL-31Rapolypeptide or an antibody that immunospecifically binds to a IL-31Rapolypeptide.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” are encoded by a variableregion gene at the NH2-terminus and a kappa or lambda constant regiongene at the COOH-terminus. Full-length immunoglobulin “heavy chains”,are similarly encoded by a variable region gene and one of the otheraforementioned constant region genes (about 330 amino acids). Heavychains are classified as gamma, mu, alpha, delta, or epsilon, and definethe antibody's isotype as IgG (including IgG1, IgG4), IgM, IgA, IgD andIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. (See generally, Fundamental Immunology (Paul, W., ed., 2nded. Raven Press, N.Y., 1989), Ch. 7 (incorporated by reference in itsentirety).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (See, Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and Chothia and Lesk, 1987,J. Mol. Biol. 196: 901-917) (both of which are incorporated herein byreference). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. Thus, a “humanframework region” is a framework region that is substantially identical(about 85% or more, usually 90-95% or more) to the framework region of anaturally occurring human immunoglobulin. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDR's. TheCDR's are primarily responsible for binding to an epitope of an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes and antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and(Fab′)₂, as well as diabodies, linear antibodies, multivalent ormultispecific hybrid antibodies (as described above and in detail in:Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in singlechains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85 5879-5883(1988) and Bird et al., Science, 242:423-426 (1988), which areincorporated herein by reference). (See, generally, Hood et al.,“Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,Nature, 323:15-16 (1986), which are incorporated herein by reference).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding. Single chain antibodies are discussedin detail by Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315(1994); see also International Patent Application Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures ofwhich are incorporated by reference for any purpose. In specificembodiments, single-chain antibodies can also be bi-specific and/orhumanized.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)−V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)−C_(H1)−V_(H)−C_(H1)) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least thevariable domains of the immunoglobulin heavy and light chains. Animmunologically functional immunoglobulin fragment of the invention iscapable of binding to a ligand, preventing binding of the ligand to itsreceptor, interrupting the biological response resulting from ligandbinding to the receptor, or any combination thereof.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single clone, including any eukaryotic, prokaryotic, or phageclone, and not the method by which it is produced.

The present invention is based in part upon the discovery that thesubunits of the heterodimeric receptor which binds IL-31, e.g. IL-31Raand OSMRb, are expressed on neural cells such as dorsal root ganglioncells. Thus the present invention encompasses the use of IL-31Ra andOSMRb as antagonis, which inhibiting pain and inflammation and thesymptoms of inflammatory bowel disease, Crohn's disease, pruritis, andneurogenic pain and sensitization by binding to IL-31 such that signaltransduction is inhibited. The present invention also encompases the useof IL-31 agonists in improving sensitization through stimulation of thedorsal root gangion cells.

IL-31 is the HUGO name for a cytokine that has been previously describedas Zcyto17rlig in a published U.S. patent application (See publishedU.S. patent application number 20030224487, U.S. patent application Ser.No. 10/352,554, filed Jan. 21, 2003, now issued U.S. Pat. No. 7,064,186;Sprecher, Cindy et al., 2003, incorporated herein by reference). Theheterodimeric receptor for IL-31, comprises a heterodimer formed betweenIL-31Ra and OncostatinM receptor beta (OSMRb). IL-31Ra is the HUGO namefor a protein called zcytor17 in commonly-owned U.S. published patentapplication number 20030215838, U.S. patent application Ser. No.10/351,157, filed Jan. 21, 2003, herein incorporated by reference. Thepolynucleotide and polypeptide sequences for human IL-31 are shown inSEQ ID NOs: 1 and 2, respectively. The polynucleotide and polypeptidesequences for murine IL-31 are shown in SEQ ID NOs: 3 and 4,respectively. As used herein the term, IL-31 shall mean zcytor17lig asused in U.S. patent publication number 20030224487, as shown above.IL-31Ra has been previously described in commonly-owned U.S. patentapplication Ser. No. 09/892,949 filed Jun. 26, 2001, which is hereinincorporated by reference.

The amino acid sequence for the OSMR, and IL-31RA receptors indicatedthat the encoded receptors belonged to the Class I cytokine receptorsubfamily that includes, but is not limited to, the receptors for IL-2,IL-4, IL-7, Lif, IL-12, IL-15, EPO, TPO, GM-CSF and G-CSF (for a reviewsee, Cosman, “The Hematopoietin Receptor Superfamily” in Cytokine 5(2):95-106, 1993). The zcytor17 receptor is fully described incommonly-owned PCT Patent Application No. US01/20484 (WIPO publicationNo. WO 02/00721; herein incorporated by reference).

The present invention includes the use of anti-IL-31Ra and anti-OSMRbmolecules , including antagonists, antibodies, binding proteins,variants and fragments, having anti-IL-31 activity. The inventionincludes administering to a subject the anti-IL-31Ra and or anti-OSMRbmolecule and contemplates both human and veterinary therapeutic uses.Illustrative veterinary subjects include mammalian subjects, such asfarm animals and domestic animals.

The native polynucleotide and polypeptide sequences for the “long” formof IL-31RA are shown in SEQ ID NOs:5 and 6, respectively. The nativepolynucleotide and polypeptide sequences for the “short” form of IL-31RAare shown in SEQ ID NOs:7 and 8, respectively. Additional truncatedforms of IL-31RA polypeptide appear to be naturally expressed. Bothforms encode soluble IL-31RA receptors. The “long” soluble IL-31RApolynucleotide and polypeptide sequences are shown in SEQ ID NOs:9 and10, respectively. The “short” soluble IL-31RA polynucleotide andpolypeptide sequences are shown in SEQ ID NOs:11 and 12, respectively.The native polynucleotide and polypeptide sequences for mouse IL-31 RAare shown in SEQ ID NOs:13 and 14, respectively. The nativepolynucleotide and polypeptide sequences for human OSMRbeta are shown inSEQ ID NOs:15 and 16, respectively. See PCT applications WO 02/00721 andWO 04/003140, both of which are incorporated by reference. Thus,examples of antagonists of neurogenic inflammation and stimualationinclude a soluble m-31Ra subunit that binds IL-31, a soluble multimer ofthe IL-31Ra subunit, such as a homodimer, and a soluble heterodimer ofthe IL-31Ra and OSMRb that binds IL-31. See Example 9 for a constructionof a soluble heterodimer of IL-31Ra and OSMRb. Other solubleheterodimeric constructs and protein can be built and are describedherein.

IL-31Ra and OSMRb antagonists include molecules that bind IL-31,including, soluble receptors, variants, fragments or derivativesthereof, or antibodies to IL-31 Ra and/or OSMRb, that inhibit, limit,reduce, minimize, prevent, or neutralize the effect of IL-31 has onbinding its cognate receptor.

In situ expression analysis revealed that IL-31RA and OSMRbeta areexpressed in the spinal cord and dorsal root ganglion cells in humans.See Example 1. Therefore, IL-31 molecules, their agonists, orantagonists play a role in the maintenance of neurons and neurogenicinflammation and stimulation. This indicates that IL-31Ra and OSMRbagonists, antagonists can be used to treat a variety ofneurodegenerative diseases such as amyotrophic lateral sclerosis (ALS),Alzheimer's disease, Huntington's disease, Parkinson's disease,peripheral neuropathies, and demyelinating diseases including multiplesclerosis. The tissue specificity of IL-31RA and OSMRb suggests thatIL-31 may be a growth and/or maintenance factor in the spinal cord andbrain which can be used to treat spinal cord, brain or peripheralnervous system injuries.

Methods of measuring the ability of IL-31 to stimulate pain are known toone of skill in the art. For example, dorsal root gangion cells can beisolated and cultured. See Voilley, N. et al., J. Neurosci.,27(20):8026-8033, 2001. For example, dorsal root ganglion cells areprepared from Wistar adult male (5-7 weeks) and newborn rats by 0.1%collagenase dissociation and plating on collagen coated P35 dishes inDMEM plus 5% fetal calf serum. Similarly methods of isolating dorsalroot ganglion cells are described by Steinhoff, M. et al. (SeeSteinhoff, M. et al., Nature Medicine, 6(2):151-157, 2000). Briefly,dorsal root ganglion cells are minced in cold Dulbeccos' modifiedEagle's Medium (DMEM) and incubated in DMEM containing 0.05 mg/mltrypsin, 1 mg/ml collagenase, and 0.01 mg/ml DNAse I for 45-60 minutesat 37 degrees C. SBTI is added to neutralize trypsin and the suspensionis centrifuged at about 1,000 g for 1 min. Neurons in the pellet aresuspended in DMEM containg 10% fetal bovine serum, 5 ng/ml nerve growthfactor, 2 mM glutamine, 1 mg/ml penicillin/streptomycin and DNAse I, andplated on glass coverslips coated with Matrigel. Neurons are culturedfro 3-5 days before use. Expression of IL-31Ra at the plasma membranesis verified by immunofluorescence using an antibody.

To measure the effect antagonists of IL-31Ra and/or OSMRb on IL-31stimulation of dorsal root ganglion, intracellular calcium ionconcentration is measured in the cultured neurons as described bySteinhoff et al., supra. The neurons are incubated in Hank's balancedsalt solution, 20 mM HEPES, pH 7.4 containing 5 uM Fura-2/AM (MolecularProbes, Eugene, Oregon) for 1 h at 37 degrees C. Coverslips are washed,mounted in a chamber (1 ml volume) on a Zeiss 100 TV inveted microscopeand observed using a Zeiss x40 Fluar objective. Fleuorescence ismeasured at 340 nm and 380 nm to allow determination of calcium. Cellsare exposed to IL-31 with and without other sensitization agents, andinhibition in the presence of IL-31RA and/or OSMRb antagonists ismeasured.

To measure the ability of an IL-31RA and/or OSMRb antagonist on IL-31binding to its cognate heterodimeric receptor on dorsal root gangion, orneural cells in general, on pain several mediators of pain can bemeasured, such as for example, but not limited to, prostaglandins,substance P, CGRP, galanin, Neuropeptide Y, histamine, bradykinin,cannabinoids, and mediators of the arachinoid acid pathway.

In addition to the above in vitro methods to measure the ability ofantagonists to IL-31Ra and OSMRb on pain-inducing effect of IL-31 onneural cells, several in vivo models are also useful. See, for example,Honore, P. et al., Neuroscience, 98(3):585-598, 2000. This articledescribes several models for inflammatory pain, neuropathic pain andcancer pain. For example, one model measures the effect of an antagonistto IL-31Ra, such as a subcutaneous injection of IL-31, with and with outthe antagonist molecule, into the plantar surface of the hindpaw of amouse. The mouse is euthanized 3 days after injection peripheral edemais measured. The effect of the IL-31Ra or OSMRb antagonist molecule toinhibit, limit, minimize, reduce, prevent, or neutralize the edema ismeasured. Additonal in vivo models are spinal nerve ligation, sciaticnerve transaction, sarcoma-induced bone cancer, behavioral analysis, andeffects of morophine.

Another mouse model of pain is mechanical allodynia. See for example,Sweitzer, S. M. et al., J. Neuroimm., 125:82-93, 2002. Briefly, rats ormice are tested for mechanical allodynia with 2- and/or 12-g von Freyfilaments. First the animals are acclimated to the procedure andbaseline measurement are taken. The IL-31 is administered in varyingamounts. Allodynia is characterized as an intense withdrawl of the pawto a normally non-noxious stimuli in response to IL-31 administration.Comparision is made with and without administration of the IL-31Ra and/or OSMRb antagonists molecule(s).

A proinflammatory neuropeptide, Substance P (SP), is made the dorsalganglia and then transported to the periphery by nociceptive nerves Aand C (15). SP can induce itch by releasing histamine from the mast cellgranules. In the skin, SP can also cause erythema, edema and neurogenicinflammation releasing histamine, IL-1, prostaglandins and lysosomalenzymes but is quickly degraded in the dermis (16). The prior oraladministration of antihistamines inhibits the pruritus caused by SP.Capsaicin obtained from hot pepper applied locally depletes SP fromcutaneous nerves, and so diminishes pruritus. As the receptor subunitsfor IL-31 are expressed in the dorsal root ganglion cells,administration of the IL-31Ra and/or OSMRb antagonist molecules candecrease the stimulation of these cells and may decrease Substance Pthat may be induced by IL-31 administration.

The binding of IL-31 to its receptor, i.e., IL-31RA and OSMR beta, ondorsal root ganglion cells can stimulate the somatosensory system, whichconsists of various sensory receptors responsible for sensations such aspressure, touch, temperature, pain, itch, tickle, tingle, and numbness.The binding of IL-31 to its cognate receptor can result in neurogenicinflammation and stimulation, which may lead to release of additionalfactors that induce neurogenic stimulus. One group of factors thatmediate pain is the prostaglandins, which also contribute to localinflammation. Thus, an IL-31 antagonist may have benefit in acuteinflammatory pain commonly treated with NSAIDs, such as myalgia,headache, joint pains from acute injuries or chronic pain such as thatcaused by osteoarthritis. Such neurogenic stimulus can be the result ofinflammation caused by, for example, autoimmune reactions, such asallergy, viral infection, such as varicella, and injury, such as burn ortrauma. Thus, antagonists that interfere with signal transductioninduced by the binding of the IL-31 ligand to its cognate receptor canbe useful in reducing, limiting, preventing, or minimizing neurogenicinflammation and the stimulation of the somatosensory system. As such,antagonists of IL-31-induced signal transduction in dorsal root ganglioncells can be used to treat pain, itch, tingling, associated withautoimmune diseases, viral infection, and trauma. Moreover, sinceneurogenic inflammation can result in a hypersensitivity of the nerveafter the initial insult, antagonists of IL-31Ra and/or OSMRb can beeffective treatment of symptoms. For example, some shingles patientsexperience the sensory symptoms of pain and/or itch long after the viralinfection has been cleared or minimized. The neuralgia that accompaniesacute herpes zoster, and postherpetic neuralgia are likely due toinflammation of the dorsal root ganglia and trigeminal ganglia, whereviral antigens attract T cells and other inflammatory cells. Longlasting pain may result from persistent inflammation of the dermatomefollowing a robust antiviral response. Consequently, the level or stageof viral infection may not be representative of the sensory perceptionof the subject. Thus, the beneficial effect of antagonizingIL-31-induced signal transduction may extend beyond the immediate stateof viral infection or trauma.

Neuropathy and sensory deficiency involve pain and loss of sensitivity,and can be related to such diseases as, atopy, diabetes, multiplesclerosis, and hypertension, for example. As EL-31RA and OSBRbeta areproteins that are expressed in the spinal cord and dorsal root ganglioncells, antagonists of IL-31Ra and/or OSMRb may be useful to treat painand sensory deficiencies. For example, the IL-31Ra and/or OSMRbantagonists can be delivered topically, subcutaneously, centrally, orsystemically, to treat diabetic peripherineuropathy, postherpaticperipheral neuropathy, as well as pain, in general, including pain as asymptom in burn patients.

Burn injuries cause intense and prolonged pain that is intensified whenthe wound dressing is changed. Frequent dressing changes are necessaryto prevent infection and aid healing. The amount of pain experienced bypatients during wound care remains a worldwide problem for burn victimsas well as a number of other patient populations. When patients are atrest pain associated with burn can be treated with opioids, which havesome unwanted effects. However, during wound care such as daily bandagechanges, wound cleaning, staple removals etc., opioids are not enough,with a majority of burn patients reporting severe to excruciating painduring wound care.

Since both members of the heterodimer for IL-31, i.e., IL-31RA andOSMRbeta are expressed in dorsal root ganglion cells, an antagonist toIL-31Ra and/or OSMRb, such as a neutralizing antibody is useful toprevent, minimimize, limit and/or treat pain, including pain associatedwith burn or neuropathy. In vivo models mimicking burn are well known toone skilled in the art.

Persistent pain can provoke hyperplasia such that less than the originalstimulus can cause increased pain, also called allodynia. As both theIL-31RA and OSMR beta subunits are expressed on dorsal root gangioncells, an antagonist to IL-31Ra and/or OSMRb induced signal transductionin neuronal cells bearing these subunits can help to mitigate symptomsof allodynia.

Polypeptides of the present invention, such as IL-31Ra and/or OSMRb, aswell as agonists, fragments, variants and/or chimeras thereof, can alsobe used to increase sensitization in mammals. For example, IL-31polypeptides of the present invention, including agonists, can be usedto increase sensitization (pain, heat, or mechanical) when deliveredlocally or topically, systemically, or centrally and measured in anymodels or experiments known to one skilled in the art and/or describedherein. Also, the polypeptides of the present invention can beadministered to enhance the sensitivity of spinal and neuronal cells inorder to improve the function of the surviving neurons toneurotransmitters and therefore might be effective in Parkinson's orAlzheimers disease, as well as paralysis.

Similarly, where a patient has an increased sensitization to pain,antagonists to IL-31Ra and/or OSMRb can be used to decrease thesensation of pain in a patient with neuropathy. For example a patientwith diabetic neuropathy and postherpatic neuropathy, have chronic,enhanced pain, the antagonist to IL-31Ra and/or OSMRb may be useful tolimit, prevent or decrease the pain.

As a receptor for a protein that is proinflammatory, the presence ofIL-31RA and OSMRbeta in the spinal cord and dorsal root ganglionindicate that antagonists of IL-31Ra and/or OSMRb can be used to reduceinflammation in these tissues. Thus, conditions such as meningitis maybenefit from administration of the antagonists, including antibodies.

Diseases which involve neurogenic inflammation and stimulation and canbenefit from antagonizing IL-31 induced pain in neuronal tissues,including dorsal root ganglion cells include: chronic pain, migraines,arthritis, osteoarthritis, rheumatoid arthritis, polyneuropathy,diabetic peripheralneuropathy, pain subsequent to nerve severence (eg.post-surgical pain), inflammatory conditions that involve a neurogenicpain-producing component, such as inflammatory bowel disease, nephritis,certain metastic carcinomas, and inflammation of the blood vessels.These diseases can also be treated by an antagonist of IL-31 inducedsignal transduction. In addition, skin conditions, including radiationirritation and burns, chemical burns, multiple chemical sensitivity,prickly heat, rhinitis, thermal burns, sunburn, reddening of the skinand chemically induced lesions, and acute allergic reactions such asacute asthma attack and inflammation of the lung caused by chemicalexposure, and hives as well as conjunctivitis and gum disease can betreated with IL-31Ra and/or OSMRb antagonists. Additionally,scapuloperoneal syndromes are heterogeneous neuromuscular disorderswhich are characterized by weakness in the distribution of shouldergirdle and peroneal muscles. Both neurogenic (scapuloperoneal spinalmuscular atrophy, SPSMA) and myopathic (scapuloperoneal musculardystrophy, SPMD) scapuloperoneal syndromes have been described. Thechromosomal locus for SPMD has recently been assigned to chromosome 12q,which is the same locus as for IL-31. Thus, IL-31Ra and/or OSMRbantagonists can be used to treat these diseases.

In the United States approximately 500,000 people suffer frominflammatory bowel disease, which can involve either or both the smalland large bowel. Ulcerative colitis and Crohn's disease are thebest-known forms of inflammatory bowel disease, and both are categorizedas “idiopathic” inflammatory bowel disease because the etiology for themis unknown.

Crohn's disease can involve any part of the gastrointenstinal tract, butmost frequently involves the distal small bowel and colon. Inflammationcan produce anything from a small ulcer over a lymphoid follicle to adeep fissuring ulcer to transmural scarring and chronic inflammation.Although the etiology is unknown, infectious and immunologic mechanismshave been proposed. Symptoms are variable and can include diarrhea,fever, and pain, as well as extra-intestinal manifestations ofarthritis, uveitis, erythema nodosum, and ankylosing spondylitis.

The traditional approach to treating inflammatory bowel disease isimmunosuppression with azathioprine (see, for example, Rutgeerts, J.Gastroenterol. Hepatol. 17(Suppl.):S176-85 (2002)). More recently, thechimeric monoclonal anti-tumor necrosis factor antibody, infliximab, hasbeen used to target specific pathogenic disease mechanisms, and allowsthorough suppression of the disease process and healing of the bowel inthe long term. However, this therapy is associated with problems ofimmunogenicity. The formation of antibodies to infliximab interfereswith efficacy and is associated with infusion reactions.

Irritable bowel syndrome (IBS) is a chronic functional gastrointestinaldisorder. It is a heterogeneous condition characterized by a variety ofbowel symptoms including abdominal pain and bloating which are usuallyassociated with altered bowel habit (Collins et al, 2001). It isestimated that between 12 and 20% of the U.S. population suffer fromthis condition. Differing criteria have been proposed for defining lBS,including the Manning criteria (Manning et al, 1978), the Rome criteria(Thompson et al, 1992), and most recently Rome II (Thompson et al.,1999). Research reports on LBS frequently classify patients with IBSinto the two subtypes of constipation predominant (CON) and diarrheapredominant (DIA) and sometimes include a third subtype of alternatingpattern (ALT).

Anti-IL-31Ra and/or anti-OSMRb molecules, antagonists, antibodies,binding proteins, variants and fragments, are useful in treating,detecting, and pain associated with Inflammatory Bowel Disease (IBD) andIrritable Bowel Syndrome (IBS).

Inflammatory Bowel Disease (IBD) can affect the colon and/or rectum(Ulcerative colitis), or the small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. Potentialtherapeutics include anti-IL-31Ra and/or OSMRb molecules, including,anti-IL-31Ra and/or OSMRb antibodies, other binding proteins, variants,fragments, chimeras, and other IL-31Ra and/or OSMRb antagonists. Thesemolecules could serve as a valuable therapeutic to reduce inflammationand pathological effects in IBD and related diseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers form,releasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress. Thus, theanti-IL-31 molecules of the present invention can be useful to treat andor detect UC.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanied by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of IL-31Ra and/or OSMRb antagonists or bindingpartners to these TNBS or DSS models can be used to measure theamelioration of symptoms and alter the course of gastrointestinaldisease. IL-31 or may play a role in the inflammatory response and painassociated with colitis, and the neutralization of IL-31 activity byadministrating antagonists is a potential therapeutic approach for IBD.

Irritable Bowel Syndrome is one of the most common conditions in thegastrointestinal clinic. Yet, diagnosis and treatment for IBS remainlimited. As the expression of IL-31 and IL-31RA1 have been correlatedwith upregulaiton of Crohn's disease (See Example 5). IL-31Ra and/orOSMRb antagonists, including anti-IL-31Ra and/or OSMRb antibodies, otherbinding proteins, variants, fragments, chimeras, and other IL-31Raand/or OSMRb antagonists are useful in reducing symptoms and treatmentof the disease.

The administration of IL-31Ra and/or OSMRb antagonists or bindingpartners to a patient with IBD or IBS can be used to ameliorate symptomsand alter the course of gastrointestinal disease. IL-31 may play a rolein the inflammatory response in colitis, and the neutralization of IL-31activity by administrating antagonists is a potential therapeuticapproach for IBD and/or IBS.

For disorders related to IBS and IBD, clinical signs of improvedfunction include, but are not limited to, reduction in pain, crampingand sensitivity, reduction in diarrhea and improved stool consistency,reduced abdominal distension, and increased intestinal transit.Improvement can also be measured by a decrease in mean Crohn's DiseaseActivity Index (CDAI). See Best. W. et al., Gasttoenterology 70: 439-44,1976. Additonally, improved function can be measured by a quality oflife assessment as described by Irvine et al. (Irvine, E. et al.,Gasttoenterology 106: 287-96, 1994.

Animal models of irritable bowel syndrome are described by Mayer andCollins. Gastroenterol. 122:2032-2048 (2002). These models can bedivided into those that are mediated primarily by CNS-directedmechanisms (“Stress Memory” models) and those with primary gut-directedetiologies (“Pain Memory” and “Immune Memory” models). In one model,animals are surgically prepared with electrodes implanted on theproximal colon and striated muscles, and catheters implanted in lateralventricles of the brain. Rectal distension is performed by inflation ofa balloon rectally inserted, and the pressure eliciting a characteristicvisceromotor response is measured. A test compound, such as IL-31Raand/or OSMRb antagonist and/or variants or antagonists, is administeredvia the appropriate route (p.o., i.p., s.c., i.v., or i.m.) and at theappropriate time (i.e. ˜20 min, if i.p. or i.c.v.) prior to distention.Test compound is evaluated for its ability to affect colonic motility,abdominal contractions, and visceral pain.

Additionally, disorders associated with inflammation of the intestinecan be treated with the IL-31Ra and/or OSMRb antagonists such asfragments, agonists and antagonists thereof described herein. Forexample, Irritable Bowel Syndrome (IBS) is characterized by a very broadspectrum of symptoms (pain; bouts of diarrhea and/or constipation;abnormal gastrointestinal motility). It is difficult to pinpoint theetiology, and may have components related to stress, genetics, and/orinflammation. Simiarly, the anti-IL-31Ra and/or OSMRb molecules of thepresent invention, including antibodies and binding partners, can beused to treat Inflammatory Bowel Disease, (including colitis and Crohn'sdisease). IBD is more serious than IBS, and is characterized bydiarrhea, pain, and malnutrition. Patients with IBD often have increasedrisk of gastrointestinal cancer.

Gastrointestinal motor activity can be measured in a dog model asfollows: Dogs are anesthetized and the abdominal cavity opened.Extraluminal force transducers (sensor to measure contraction) aresutured onto five (5) sites, i.e., the gastric antrum, 3 cm proximal tothe pyloric ring, the duodenum, 5 cm distal to the pyloric ring, thejejunum, 70 cm distal to the pyloric ring, the ileum, 5 cm proximal tothe ileum-colon junction, and the colon, 5 cm distal to the ileum-colonjunction. The lead wires of these force transducers are taken out of theabdominal cavity and then brought out through a skin incision madebetween the scapulae, at which a connector is connected. After theoperation, a jacket protector is placed on the dog to protect theconnector. Measurement of the gastrointestinal motor activity is startedtwo weeks after the operation. For ad libitum measurement, a telemeter(electrowave data transmitter) is connected with the connector todetermine the contractive motility at each site of the gastrointestinaltract. The data is stored in a computer via a telemeter for analysis. Atest compound, such as IL-31 antagonist is administered via theappropriate route (p.o., i.v., i.p., s.c., i.m.) at the appropriate timepoint to assess its ability to affect gastrointestinal motor activity.This can be performed in normal dogs or dogs in whichgastroparesis/ileus has been induced. The above method is a modificationof those in Yoshida. and Ito. J. Pharmacol. Experiment. Therap. 257,781-787 (1991) and Furuta et al. Biol. Pharm. Bull. 25:103-1071 (2002).

IL-31 may be a trigger for reactivation of latent viral infections, suchas varicella infection. In primary varicella zoster virus (VZV)infection, the T cells most likely to be infected by varicella zostervirus are CD4 positive memory T cells expressing CLA and CCR4. These areskin-homing T cells, which may enhance cell-associated viremia and thetransport of infectious virus to the skin and DRG. These cells are alsothe primary producers of IL-31. Thus, IL-31 in primary VZV infection maycontribute to the itch/pain involved in the skin lesions. Reactivationof latent virus in DRG induces VZV-specific T cell responses, whichcontribute to the neurogenic inflammation. Skin-homing T cells are mosteasily infected with VZV, and in vivo transfer of virus from T cells toDRG has been observed. Postherpetic neuralgia is one of the majorcomplications of herpes zoster caused by the reactivation ofvaricella-zoster virus and is characterized by severe pain. SeeSato-Takeda, M. et al., Anesthesiology. 2006 104(5):1063-9, hereinincorporated by reference. This reference also teaches a mouse model ofpostherpetic pain, which corresponds to postherpetic neuralgia. Briefly,BALB/c mice (MHC haplotype: H-2), C57BL/6 mice (MHC haplotype: H-2), andBALB/b mice, a congenic BALB/c strain with H-2, are transdermallyinoculated on the hind paw with Herpes simplex virus type I.Unilaterally zosteriform skin lesion and pain-related responses (acuteherpetic pain) are caused, and some mice show pain-related responses(postherpetic pain) after the cure of skin lesions. Herpes simplex virustype I antigen and CD3-positive cells are immunostained in the dorsalroot ganglion in the acute phase. See also Argoff, C. E., et al., J PainSymptom Manage. 2004 October; 28(4):396-411, herein incorporated byreference. Antagonists to IL-31Ra and/or OSMRb can be used to limit orprevent reactivation of viral infections with varicella.

Mouse models for experimental allergic encephalomyelitis (EAE) has beenused as a tool to investigate both the mechanisms of immune-mediateddisease, and methods of potential therapeutic intervention. The modelresembles human multiple sclerosis, and produces demyelination as aresult of T-cell activation to neuroproteins such as myelin basicprotein (MBP), or proteolipid protein (PLP). Inoculation with antigenleads to induction of CD4+, class H MHC-restricted T-cells (Th1).Changes in the protocol for EAE can produce acute, chronic-relapsing, orpassive-transfer variants of the model (Weinberg et al., J. Immunol.162:1818-26, 1999; Mijaba et al., Cell. Immunol. 186:94-102, 1999; andGlabinski, Meth. Enzym. 288:182-90, 1997). Administration of IL-31antagonists or other soluble and fusion proteins may be useful toameliorate symptoms and alter the course of disease.

Antagonists to IL-31-induced signal transduction in dorsal root gangioncells, such as anti-IL-31Ra and/or anti-OSMRb can be useful to treatpruritus uraemicus; pruritis from hepatitis, hepatic failure, orcholestasis; from scabies or athletes's foot; from pruritis associatedwith pregnancy; from pruritis in dualysis patients; and from pruritisfrom anaesthasia and psychological disorders as follows.

Pruritus uraemicus or renal itch is an often intolerable symptom ofchronic renal insufficiency (Blachley J D, Blankenship D M, Menter A etal. Uremic pruritus: skin divalent ion content and response toultraviolet phototherapy. Am J Kidney Dis 1985; 5: 237-41.) beingpresent in about 13% of the cases; secondary skin lesions due toscratching can be seen. It is even more common in patients undergoingperitoneal dialysis or hemodialysis (Murphy M, Carmichael A J. Renalitch. Clin Exp Dermatol 2000; 25: 103-6.); it can be localized orgeneralized. Itching is not present in acute renal failure. Thetreatment of renal pruritus is based on intensive and efficient dialysisto remove pruritogenic substances from the blood, and on the use ofnon-complement-activating membranes. One can also use UV therapy,emollient ointments, activated charcoal, cholestyramine (4 grams twice aday), phosphate binding agents. Sometimes parathyroidectomy isnecessary.

Pain antagonizes itch. See, for example, Ward, L. et al., Pain64:129-138, 1996. As such a mediator of pain, such as an IL-31Ra and/orOSMRb antagonist can be used to treat pain associated with itch, therebyameliorating not only the itch, or scratching behavior, but also theassociated pain.

Pruritus is a well-recognized manifestation among patients with liverdiseases and intrahepatic or posthepatic cholestasis. Hepatic diseasesleading to pruritus include primary biliary cirrhosis, B and C viralhepatitis, primary sclerosing cholangitis, carcinoma of bile ducts,alcoholic cirrhosis, autoimmune hepatitis and others. The pruritus isgeneralized and more intense on hands, feet and around tight-fittingclothes, while face, neck and genital area are rarely involved.

Generalized pruritus is present in 1-8% of pregnant women. Pruritusgravidarum can be differentiated from pruritic dermatoses in pregnancy,such as pemphigoid gestationis (herpes gestationis), papular andpruritic dermatosis of pregnancy and others. Pruritus gravidarummanifests without any rash mostly in the third trimester of pregnancy,but it may also appear earlier, firstly on the abdomen and then becomesgeneralized. This symptom usually tends to be worse at night anddisappears after delivery (within 1-4 weeks). Probably it is associatedwith intrahepatic cholestasis, as there is an increase of gamma GT andalkaline phosphatase, and sometimes also of direct bilirubin level inthese patients. Pruritus is more frequent in multiple pregnancies andcan recur in subsequent pregnancies or during the use of oralcontraceptives. Additonally, pruritic urticarial papulas and plaques ofpregnancy (PUPP), the most common dermatosis associated with pregnancy,does not respond to antihistamines and often persists beyondparturition.

Some hematological disorders are known to be associated with pruritus.In polycythemia rubra vera with overproduction of all threehematopoietic cell lines, patients typically experience severe itchlocated on the trunk, but sparing the face, hands and feet, a fewminutes after contact with warm water. Water-induced itching (aquagenicpruritus, or bath itch) can be present in 70% of the patients. The itchcan last for about 15 minutes to one hour, and be so severe that thepatients refuse to bathe. In the last decades pruritus has beendescribed in patients with graft versus host reactions after bone marrowtransplantation.

Chronic delivery of IL-31 induces pruritis and alopecia in mice followedby the development of skin lesions resembling dermatitis suggesting thatIL-31 may induce itching. See See Dillon S. R., et al., Nat Immunol: 5,752 (2004). The involvement of IL-31 was tested in induction of the itchresponse by two methods as shown in Example 2: (i) capsaicin treatmentof IL-31-treated mice and (ii) IL-31 treatment of Tacl knockout mice,which have significantly reduced nociceptive pain responses because oflack of expression of neuropeptides. In addition, whether neutralizationof IL-31 in IL-31 treated mice could prevent pruritis and alopecia wastested in Example 2.

NC/Nga Mice spontaneously develop AD-like lesions that parallel human ADin many aspects, including clinical course and signs, histophathologyand immunopathology when housed in non-specified pathogen-free (non-SPF)conditions at around 6-8 weeks of age. In contrast, NC/Nga mice keptunder SPF conditions do not develop skin lesions. However, onset ofspontaneous skin lesions and scratching behaviour can be synchronized inNC/Nga mice housed in a SPF facility by weekly intradermal injection ofcrude dust mite antigen. See Matsuoka H., et al., Allergy: 58, 139(2003). Therefore, the development of AD in NC/Nga is a useful model forthe evaluation of novel therapeutics for the treatment of AD.

In addition to the NC/Nga model of spontaneous AD, epicutaneoussensitization of mice using OVA can also be used as a model to induceantigen-dependent epidermal and dermal thickening with a mononuclearinfiltrate in skin of sensitized mice. This usually coincides withelevated serum levels of total and specific IgE, however no skin barrierdysfunction or pruritus normally occurs in this model. See Spergel J.M., et al., J Clin Invest, 101: 1614, (1998). This protocol can bemodified in order to induce skin barrier disregulation and pruritis bysensitizing DO11.10 OVA TCR transgenic mice with OVA. Increasing thenumber of antigen-specific T cells that could recognize the sensitizingantigen may increase the level of inflammation in the skin to inducevisible scratching behaviour and lichenification/scaling of the skin.

Both the NC/Nga spontaneous AD model and the OVA epicutaneous DO11.10model can be used to measure expression of IL-31 and IL-31RA in AD, aswell as the ability of the antagonists described herein to inhibit,reduce, or neutralize the effects of IL-31. The antagonists describedherein are useful to inhibit scratching associated with dermatitis andpruritic diseases including atopic dermatitis, prurigo nodularis, andeczema. In AD, the scratching behavior provoked by intensely itchy skinis believed to aggravate disease by breaking down skin barrier functionsand activating keratinocytes, leading to chemokine production andincreased inflammation. Many clinicians view AD as a self-propagatingcycle, since lesions formed by frequent scratching are subject toinfection and further antigen stimulation. The fact that patients withnear total involvement of body surface area may have unaffected skin inregions that are hard to scratch lends credence to this hypothesis. Bypreventing pruritis, administration of antagonists of IL-31Ra and/orOSMRb can be effective in treating pruritic disease by decreasingIL-31-induced keratinocyte activation and neurological stimulation, thusbreaking the link between inflammation and pruritis,. The reduction inpruritis could also decrease secretion of neurostimulatory factors andreduce the inflammation and excoriations associated with constantscratching, leading to an improvement in disease scores and/or a longerduration between disease flares. An inhibition, reduction, or preventionof scratching, alone, can be effective in treating pruritic diseasesincluding, but not limited to, atopic dermatitis, prurigo nodularis, andeczema, since cessation of scratching will stop progression ofdermatitis, the development of which is dependent on scratching.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)2 and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication No.WO 98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-IL-31 antibodies herein bind to a IL-31polypeptide, peptide or epitope with an affinity at least 10-foldgreater than the binding affinity to control (non-IL-31) polypeptide. Itis preferred that the antibodies exhibit a binding affinity (Ka) of 106M-1 or greater, preferably 107 M-1 or greater, more preferably 108 M-1or greater, and most preferably 109 M-1 or greater. The binding affinityof an antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad.Sci. 51: 660-672, 1949).

Whether anti-IL-31 antibodies do not significantly cross-react withrelated polypeptide molecules is shown, for example, by the antibodydetecting IL-31 polypeptide but not known related polypeptides using astandard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are those disclosed in the prior art, such asknown orthologs, and paralogs, and similar known members of a proteinfamily. Screening can also be done using non-human IL-31, and IL-31mutant polypeptides. Moreover, antibodies can be “screened against”known related polypeptides, to isolate a population that specificallybinds to the IL-31 polypeptides. For example, antibodies raised to IL-31are adsorbed to related polypeptides adhered to insoluble matrix;antibodies specific to IL-31 will flow through the matrix under theproper buffer conditions. Screening allows isolation of polyclonal andmonoclonal antibodies non-crossreactive to known closely relatedpolypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol.2: 67-101, 1984. Specifically binding anti-IL-31 antibodies can bedetected by a number of methods in the art, and disclosed below.

Preparation of monoclonal antibodies is well known to one skilled in theart. The purified mature recombinant human IL-31 polypeptide (amino acidresidues 27 (Leu) to 164 (Thr) of SEQ ID NO:2) or the mouse ortholog,produced from expression systems can be used to generate monoclonalantibodies.

The effect of administering the antagonists of IL-31 mediated signaltransduction can be measured in vivo by a reduction, inhibition,prevention, minimization, neutralization of inflammation, of skin ordermal thickening, of recruitment of lymphocytes, and acanthosis, forexample, and other symptoms or composites of symptoms, such as theEczema Area and Severity Index (EASI), that are evident to one skilledin the art. Additional effects could include a change or decrease in theproduction of cytokines or chemokines by lesional skin, reduction in anatopy patch test score, and decrease in release of soluble factors suchas cytokines, chemokines or neuropeptides, as measured by intradermalmicrodialysis or other methods. Assessments of degree of itch or paincan be measured using clinically approved instruments or tools such asthe Visual Analogue Scale. Frequency of scratching can be monitored bylimb movement meters, piezoelectric transducer devices attached to thefingernails, or time-lapse infrared photography or videography ofnocturnal scratching in patients. Other methods for assessing a decreasein pain or itch are evident to one skilled in the art.

Monoclonal antibodies purified from tissue culture media arecharacterized for their utility in an ELISA for the quantitativedetermination of recombinant and native human IL-31. The antibodies areselected and a quantitative assay is developed.

Monoclonal antibodies purified from tissue culture media arecharacterized for their ability to block or reduce the receptor bindingactivity (“neutralization assay”) of purified recombinant hull-31 onneural cells expressing the IL-31Ra and OSMRb. A number of“neutralizing” monoclonal antibodies are identified in this manner.Hybridomas expressing the neutralizing monoclonal antibodies to humanIL-31 described can then be deposited with the American Type TissueCulture Collection (ATCC; Manassas VA) patent depository as originaldeposits under the Budapest Treaty.

Monoclonal antibodies in tissue culture media are characterized fortheir ability to block or reduce receptor binding when grown in thepresence of the purified recombinant proteins human IL-31.

Bidnding affinity of the monoclonal antibodies can be generated.Goat-anti-Rat IgG-Fc gamma specific Antibody (Jackson) is immobilizedonto a CM5 Biacore chip. The assay is optimized to bind each mAb ontothe anti-Rat capture surface and then a concentration series of IL-31 isinjected across the mAb to see association (Ka) and dissociation (Kd).After each run, the surface is regenerated back to the anti-Rat Antibodywith 2 injections of 20 mM HCl. Data is generated for each andevaluation software (BIAevaluation software version 3.2, PharmaciaBIAcore, Uppsala, Sweden) is used to assess the kinetics of theanti-IL-31 antibody binding to the IL-31 protein

Biochemical confirmation that the target molecule, IL-31, recognized bythe putative anti-IL-31 mAbs is indeed IL-31 are performed by standardimmunoprecipitation followed by SDS-PAGE analysis or western blottingprocedures, both employing soluble membrane preparations from IL-31transfected versus untransfected Baf3 cells. The mAbs are tested fortheir ability to specifically immunoprecipitate or western blot thesoluble IL-31-muFc protein.

IL-31Ra and/or OSMRb antagonists generated by the methods describedherein can be tested for neutralization, inhibition, reduction,antagonizaiton by a variety of methods. In addition neutralization canbe tested by measuring a decrease in the production of pro-inflammatorychemokines such as TARC and MDC from keratinocyte cultures in thepresence of ligand and the monoclonal antibody. Other biomarkers, suchas MCP-1, MIP1a, TARC, MCP-1, MDC, IL-6, IL-8, I-309, SCYA19, MPIF-1,TECK, MIP-1b, SCYB13, GROa/MGSA, CTACK, SCCA1/Serpin B3, TSLP, and NT-4may also be used. Neutralization can also be measured by the in vivomodels described herein.

The bioactive antagonists or antibody conjugates described herein can bedelivered intravenously, intraarterially or intraductally,subcutaneously, topically, or may be introduced locally at the intendedsite of action.

The antagonists of the present invention can be measured for theirability to bind the IL-31 ligand as determined by any of the in vivomodels described herein, including but not limited to the NcNga model,the Ova epicutaneous model, the chronic hypersensitivity model, thechronic hapten model, the calcium flux model, the allodynia model.

Additonal models to measure the inhibitory effects of the anti-IL-31Raand/or OSMRb antagonists are known to one skilled in the art anddescribed herein are described by Umeuchi, H. et al., European Journalof Pharmacology, 518: 133-139, 2005; and by Yoo, J. et al., J.Experimental Medicine, 202:541-549, 2005.

Mouse models to measure neurogenic inflammation are known in the art.See, for example, Sweitzer, S. M., et al., J. Neuroimmunology 125:82-93; 2002, and Honore, P., et al., Neuroscience, (98): 585-598, 2000.See also, Yonehara, N. and Yoshimura M., Pain, 2001 (92/1-2): pp.259-265).

Within aspects of the invention, the invention provides methods oftreating inflammation in neuronal tissue of a mammal; methods oftreating pain in a mammal; methods of antagonizing IL-31 induced signaltransduction is dorsal root ganglion cells; methods for treatingsymptoms associated with burn; methods for treating symptoms associatedwith viral infection and for preventing reactivation of viral infection;and methods of treating pain associated with Inflammatory Bowel Disease.Within an embodiment, the Inflammatory Bowel Disease is Crohn's Disease.

Within embodiments of these aspects, the invention provides, comprisingadmixing neuronal tissue with an IL-31Ra and/or OSMRb antagonist,wherein the inflammation, pain, dorsal root ganglion signaltransduction, viral infection or reactivation, or burn tissue, or painassociated with Inflammatory Bowel Disease is reduced, limited,prevented, minimized or neutralized.

Within other embodiments, the IL-31Ra and/or OSMRb antagonist binds apolypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2from residue 27 to residue 164. Within other embodiments, the antagonistis selected from: anti-idiotype antibodies; antibody fragments; chimericantibodies; and humanized antibodies. Within another embodiment theantagonist a soluble receptor. Within other embodiments the solublereceptor comprises at least one subunit of IL-31 Ra.

Within other embodiments the neuronal tissue comprises dorsal rootganglion or spinal cord tissues.

EXAMPLES Example 1 In Situ Hybridization for IL-31RA, IL-31, and pOSMRbin Neuronal Tissues

Five human brain tissue samples and a spinal cord sample all from thesame individual, and a dorsal root ganglia (DRG) from a differentpatient were analyzed in this study.

Probes used were probes to IL-31RA, IL-31, and OSMRbeta.

Results are shown in Table 1:

TABLE 1 ISH analysis results: Tissue/probe IL-331RA pOSMRb IL-31 brainfrontal lobe − − − brain hippocampus − − − brain parietal lobe − − −brain temporal lobe, − − − brain hypothalamus − +/− − spinal cord + + −DRG + + −

Brain sections: There was no detectable amount of signal in all regionsof the brain for all three probes. There was inconsistent staining ofpOSMRb in a subset of neurons in the hypothalamus. The inconsistency maycause by very low level of pOSMRb expression that is around the level ofdetection.

Spinal cord: There was positive staining in one region of the spinalcord. The information about the possible location or orientation of thespinal cord section was unavailable. The signal appears to be in theanterior (ventral) portion of the spinal cord. The opposite side/region(also anterior) was negative. The positive signal appears to confine ina subset of larger neurons. Both IL-31RA and pOSMRb showed similarexpression patterns in this area. IL-31 was negative.

Dorsal Root Ganglion (DRG): A subset of unipolar neurons in the DRG waspositive for both IL-31RA and pOSMRb. Small satellite cells werenegative. IL-31 was negative in all cells including neurons.

Thusm an IL-31 antagonist can be useful to ameliorate symptomsassociated with neurogenic stimulation and neurogenic stimulation. Assuch the IL-31 antagonists, can be used to treat inflammation and painassociate with neural cell stimulation, such as dorsal root ganglionstimulation, and can be measured as a reduction, limitation,minimization, prevention, or neutralization of pain and inflammation.

Example 2 IL-31 Involvement in Induction of the Itch Resonse

A. Methods I (Capsaicin Treatment of IL-31 Treated Mice)

Ten week old BALB/c animals (CRL) were anaesthetized and injected with along-lasting analgesic agent, bupranorphine hydrochloride,subcutaneously at 0.1 mg/kg before injection of 0.25 ml of 4 mg,/mlsolution of capsaicin in 10% ethanol+10% Tween-80 in salinesubcutaneously into scruff of neck. Animals were kept anaesthetized forat least 30 min following neurotoxin treatment. Forty-eight hours later,14-day osmotic pumps were implanted subcutaneously for continuousdelivery of 20 ug/day of IL-31 for 14 days. Mice were monitored dailyfor 6 days for alopecia and pruritis using the following criteria: 0=noscratching, animal appears normal, 1=thinning of coat in small areas,scratching noted, 2=minor hair loss (small patches), scratching,3=moderate hair loss, scratching, and 4=severe hair loss, excessivescratching.

Results demonstrated that while non-capsaicin-treated mice showed a meanscratch/hairloss score of 2.625 following three days of IL-31 delivery,capsaicin-treated mice showed a significantly lower score of 1. Thusmice treated with capsaicin prior to IL-31 delivery showed both a delayin incidence of scratching and hairloss and a lower score in theintensity of scratching and hairloss over the six days of theexperiment. These data suggest that IL-31 does induce some neuronalcomponent that contributes to the alopecia and pruritis induced byIL-31. Therefore, neutralization of IL-31 may decrease the incidence andintensity of itch, and therefore dermatitis, in patients suffering fromskin disorders that involve itch.

B. Methods II

Mice that are homozygous null for the Tacl gene express no detectiblesubstance P or neurokinin A. These mice have significantly reducednociceptive pain responses to moderate to intense stimuli and aretherefore a useful tool for studying the contribution of tachykininpeptides to pain/itch processing and inflammatory disease states. Twelveweek old, Tad. knockout mice were implanted with 14-day osmotic pumpsdelivering lug/day of IL-31 protein and observed daily for alopecia andpruritis using the following criteria: 0=no scratching, animal appearsnormal, 1=thinning of coat in small areas, scratching noted, 2=minorhair loss (small patches), scratching, 3=moderate hair loss, scratching,and 4=severe hair loss, excessive scratching.

Results of this study show that Tac1 deficient mice were lesssusceptible to IL-31 induced scratching/hairloss compared to wildtypecontrol mice. While 100% (10/10) of wildtype mice had developed evidenceof scratching and hairloss by day 6 of IL-31 treatment, only 33.3% (2/6)Tacl deficient mice were showing signs of scratching and hairloss at thesame time-point. These data show that IL-31 induces a neuronal componentthat contributes to the scratch/hairloss phenotype in IL-31-treated miceand neutralization of IL-31 may decrease the incidence and intensity ofscratching in the context of dermatitis.

C. Methods III (Administration of IL-31 Neutralizing Antibody)

Normal female BALB/c mice (CRL) approximately 8 to 12 weeks old wereimplanted subcutaneously with 14-day osmotic pumps (Alzet, #2002)delivering 1 ug/day mIL-31. Groups of mice received intraperitoneal(i.p.) injections of rat anti-mouse IL-31 monoclonal antibody 10 mg/kg(200 ug/mouse) twice weekly starting 1 week prior to IL-31 delivery.Control groups of mice received i.p. injections of vehicle (PBS/0.1%BSA) with the identical dosing schedules. Mice were scored daily foralopecia and pruritis using the following criteria: 0=no scratching,animal appears normal, 1=thinning of coat in small areas, scratchingnoted, 2=minor hair loss (small patches), scratching, 3=moderate hairloss, scratching, and 4=severe hair loss, excessive scratching.

In all experiments, mice treated with rat anti-mIL-31 mAb had a delay inonset of symptoms of approximately 5 to 7 days and a lower overall scorefor alopecia and pruritis. All groups of mAb treated mice (regardless ofdose frequency or concentration) developed alopecia and pruritis similarto control mice by 13 day of the study. These data suggest thatneutralization of IL-31 can delay the onset of the scratch/hairlossresponse induced by IL-31.

Example 3 IL-31RA/OSMRbeta Receptor Luciferase Assay

The KZ134 plasmid was constructed with complementary oligonucleotidesthat contain STAT transcription factor binding elements from 4 genes,which includes a modified c-fos Sis inducible element (m67SIE, or hSIE)(Sadowski, H. et al., Science 261:1739-1744, 1993), the p21 SIE1 fromthe p21 WAF1 gene (Chin, Y. et al., Science 272:719-722, 1996), themammary gland response element of the β-casein gene (Schmitt-Ney, M. etal., Mol. Cell. Biol. 11:3745-3755, 1991), and a STAT inducible elementof the Fcg RI gene, (Seidel, H. et al., Proc. Natl. Acad. Sci.92:3041-3045, 1995). These oligonucleotides contain Asp718-XhoIcompatible ends and were ligated, using standard methods, into arecipient firefly luciferase reporter vector with a c-fos promoter(Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998) digestedwith the same enzymes and containing a neomycin selectable marker. TheKZ134 plasmid was used to stably transfect BaF3 cells, using standardtransfection and selection methods, to make the BaF3/KZ134 cell line.

A stable BaF3/KZ134 indicator cell line, expressing the full-lengthIL-31RA or IL-31RA/OSMRbeta receptor was constructed. Clones werediluted, plated and selected using standard techniques. Clones werescreened by luciferase assay (see B, below) using the human IL-31conditioned media or purified IL-31 protein as an inducer. Clones withthe highest luciferase response (via STAT luciferase) and the lowestbackground were selected. Stable transfectant cell lines were selected.The cell lines were called BaF3/KZ134/IL-31RA orBaF3/KZ134/IL-31RA/OSMRbeta depending on the receptors transfected intothe cell line.

Similarly, BHK cell lines were also constructed using the methoddescribed herein, and were used in luciferase assays described herein.The cell lines were called BHK/KZ134/IL-31RA orBHK/KZ134/IL-31RA/OSMRbeta depending on the receptors transfected intothe cell line.

BaF3/KZ134/IL-31RA and BaF3/KZ134/1L-31RA/OSMRbeta cells were spun downand washed in mIL-3 free media. The cells were spun and washed 3 timesto ensure removal of mIL-3. Cells were then counted in a hemacytometer.Cells were plated in a 96-well format at about 30,000 cells per well ina volume of 100 μl per well using the mIL-3 free media. The sameprocedure was used for untransfected. BaF3/KZ134 cells for use as acontrol in the subsequent assay. BHK/KZ134/IL-31RA orBHK/KZ134/IL-31RA/OSMRbeta cells were plated in a 96-well format at15,000 cells per well in 100 μl media. Parental BHK/KZ134 cells wereused as a control.

STAT activation of the BaF3/KZ134/IL-31RA, BaF3/KZ134/IL-31RA/OSMRbeta,BHK/KZ134/IL-31RA, or BHK/KZ134/IL-31RA/OSMRbeta cells is assessed usingconditioned media or purified protein. One hundred microliters of thediluted conditioned media or protein is added to the BaF3/KZ134/IL-31RA,BaF3/KZ134/IL-31RA/OSMRbeta, BHK/KZ134/IL-31RA, orBHK/KZ134/IL-31RA/OSMRbeta cells. The assay using the conditioned mediais done in parallel on untransfected BaF3/KZ134 or BHK/KZ134 cells as acontrol. The total assay volume is 200 μl. The assay plates areincubated at 37° C., 5% CO₂ for 24 hours at which time the BaF3 cellsare pelleted by centrifugation at 2000 rpm for 10 mM., and the media isaspirated and 25 μl of lysis buffer (Promega) is added. For the BHK celllines, the centrifugation step is not necessary as the cells areadherant. After 10 minutes at room temperature, the plates are measuredfor activation of the STAT reporter construct by reading them on aluminometer (Labsystems Luminoskan, model RS) which added 40 μl ofluciferase assay substrate (Promega) at a five second integration.

Example 4 Luciferase Assay on Human Transformed Epithelial Cell LinesVia Transient Infection with an Adenoviral STAT/SRE Reporter Gene

Inhibition, reduction, and/or neutralization of IL-31 activity can bemeasured by the luciferase assay. For example, human transformed celllines can be seeded in 96-well flat-bottom plates at 10,000 cell/well inregular growth media as specified for each cell type. The following day,the cells are infected with an adenovirus reporter construct, KZ136, ata multiplicity of infection of 5000. The KZ136 reporter contains theSTAT elements in addition to a serum response element. The total volumeis 100 ul/well using DMEM supplemented with 2 mM L-glutamine (GibcoBRL),1 mM Sodium Pyruvate (GibcoBRL) and 1× Insulin-Transferrin-Seleniumsupplement (GibcoBRL) (hereinafter referred to as serum-free media).Cells are cultured overnight.

The following day, the media is removed and replaced with 100 μl ofinduction media. The induction media is human IL-31 diluted inserum-free media at 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25ng/ml, 3.125 ng/ml and 1.56 ng/ml. A positive control of 20% FBS is usedto validate the assay and to ensure the infection by adenovirus issuccessful. The cells are induced for S hours at which time the media isaspirated. The cells are then washed in 50 μl/well of PBS, andsubsequently lysed in 30 μ]/well of 1× cell lysis buffer (Promega).After a 10-minute incubation at room temperature, 25 μl well of lysateis transferred to opaque white 96-well plates. The plates are then readon the Luminometer using 5-second integration with 40 μl/well injectionof luciferase substrate (Promega).

Example 5

IL-31 Analysis in Colon Tissues from Inflammatory Bowel Disease

A) IL-31 Immunohistochemistry:

A polyclonal antibody (rabbit anti-human IL-31 CEE, affinity purified to1.0 mg/ml) was used to detect human IL-31 in gastrointestinal tissuesfrom inflammatory bowel disease patients via an ABC-elite baseddetection system. Normal Rabbit Serum, Protein A purified to 1.66 mg/mlwas used as a negative control using the same protocol and antibodyconcentrations.

The protocol was as follows: ABC-HRP Elite (Vector Laboratories,PK-6100); Target Retrieval (ph 9) for 20′ steam, 20′ cooling to RT;Protein Block for 30′; Primary Ab (1:1,000-2,500) for 60′; Secondary Ab(Bi:ant-Rabbit) for 45′; ABC-HRP complex for 45′; and DAB substrate asrecommended.

In this study, a total of 19 individual GI tissues were analyzed withthe rabbit anti-human IL-31 polyclonal antibody. In this group, thereare five colon samples from normal tissue adjacent to IBD or cancertissues. Nine samples were diagnosed with Crohn's disease and five withulcerative colitis. Overall, it appears there are more cells positive inthe Crohn's samples than the normal tissues adjacent to the IBD orcancer tissues or ulcerative colitits tissues. The predominate cellswith signal in the Crohn's samples are located in the laminar propriaand submucosa, with infiltrating cells showing signal between the smoothmuscle bundles. In granulomas, many larger cells in the nodule centerare positive, however the cortex of these nodules, and Peyers patchesappear negative. The epithelium of intestinal glands is occasionallypositive. In ulcerative colitis samples, there are a small number ofscattered cells in the submucosa and infiltrating cells between smoothmuscle bundles are positive. The percentage of positive cells inulcerative colitis samples is less than that of Crohn's, but similar, orslightly higher than that of “normal” samples. Cells in the laminarpropria of ulcerative colitis are mostly negative. In summary, thisstudy demonstrates that IL31 is upregulated in Crohn's GI samples. Itappears that in this study, IL31 shows similar expression profiles inUlcerative colitis samples and “Normal” controls.

B) IL-31 In Situ Hybridization:

A subset of the tissues was also analyzed using in situ hybridization(ISH). In. ISH, IL-31 mRNA was observed in a few infiltrating cells inthe submucosa and adipose tissues. Using IHC, we observed that IL31protein stained positive in the previously mentioned cell population aswell as in cells in the laminar propria and granuloma centers. Thedifference between these two assays could be explained by assaysensitivity.

Example 6 IL-31Ra Analysis in Colon Tissues from Inflammatory BowelDisease

A) IL-31Ra Immunohistochemistry:

A polyclonal antibody (rabbit anti-human IL-31RA (version 4) CEE,affinity purified to 1.33 mg/ml) was used to detect human IL-31RA ingastrointestinal tissues from inflammatory bowel disease patients via anABC-elite based detection system. Normal Rabbit Serum, Protein Apurified to 1.66 mg/ml was used as a negative control using the sameprotocol and antibody concentrations. The rabbit anti-human IL-31RA(version 4) antibody was used at 1:2000 (665 ng/ml).

The protocol was as follows: ABC-HRP Elite (Vector Laboratories,PK-6100); Target Retrieval (ph 9) for 20′ steam, 20′ cooling to RT;Protein Block for 30′; Primary Ab (1:2,000) for 60′; Secondary Ab for45′; ABC-HRP complex for 45′; and DAB+Dako Cytomation for 10′.

In this study, a total of 19 individual GI tissues were analyzed usingthe rabbit anti-human IL-31RA (version 4) CEE antibody. In this group,there are about five colon samples from normal tissue adjacent to IBD orcancer tissues. Nine samples were diagnosed with Crohn's disease andfive with ulcerative colitis. Overall, it appears there are more cellspositive in the Crohn's samples than normal tissue adjacent to IBD orcancer tissues or ulcerative colitis tissues. The positive cells inCrohn's are primarily located in the connective tissues of submucosa.Granulomas nodules are negative. Occasionally there is weak epitheliumsignal in the Crohn's samples. There was no detectable signal in theulcerative colitis (UC) samples. A few cells in the submucosa werestained positive by IHC for the IL31RA protein.

B) IL-31Ra In Situ Hybridization:

In a previous study five tissues were studied using ISH, three of whichwere Crohn's colons. In these Crohn's tissues, IL31RA mRNA wassignificantly upregulated compared to their normal counterparts, and thesignal was localized to the cortex of granuloma nodules and manyinfiltrating cells in the connective tissues of submucosa and adiposetissue areas. Possible reasons for the discrepancy between IHC and insitu analysis includes transient mRNA expression, protein process time,IL31RA protein stability, and/or sensitivity differences between the twoassays.

Example 7 DSS-Induced Colitis Studies in EμLck IL-31 Transgenic Mice

EμLck IL-31 transgenic and non-transgenic littermate control mice weretested in a dextran sulfate sodium (DSS)-induced model of mucosalinflammation to look for potential differences in disease susceptibilityand severity. Normal mice given 2-3% DSS in drinking water developsymptoms and pathology that mimic human inflammatory bowel disease (See,Strober, Fuss and Blumberg, Arum. Rev. Immunol. 2002). Mechanistically,DSS disrupts the mucosal epithelial barrier of the large intestine,which causes subsequent inflammation. As a result of this inflammation,DSS treated mice lose body weight and develop diarrhea. Mice aremonitored for severity of colitis using a disease activity index (DAI),which is a cumulative score based on body weight, stool consistency andblood present in stool. DSS can be used to induce acute or chronic formsof colitis. Acute colitis is induced via delivery of DSS (2% or 3% inour studies) in drinking water from day 0 to day 7, while chroniccolitis is induced via delivery of DSS in the drinking water for 5 daysfollowed by a recovery phase of 7 to 12 days, before repeating the DSStreatment.

Four studies in the EμLck IL-31 transgenic mice were performed.Regardless of whether the acute or chronic model of DSS was used, theEμLck IL-31 transgenic mice lost more body weight earlier when comparedwith littermate control mice. In fact, in 3 of 4 studies the IL-31transgenic mice demonstrated significantly more weight loss compared tocontrols (p<0.001, p=0.011). Additionally, transgenic mice hadsignificantly shorter colons compared to wildtype controls (p<0.05). TheDAI score was significantly higher in IL-31 transgenic mice compared tonon-transgenic controls in a chronic colitis study (p<0.001).

To determine if systemic delivery of IL-31 could influence thedevelopment of DSS-induced colitis in normal non-transgenic mice, weimplanted animals with osmotic pumps delivering a daily dose of IL-31 orvehicle (PBS, 0.1% BSA) prior to DSS treatment. In one study, N3generation, non-transgenic mice (B6C3F2×C57BL/6) were implanted withpumps subcutaneously which delivered either 20 μg/day IL-31 or vehicleduring the course of the DSS administration. There were no differencesin weight loss, DAI score, or colon length between the IL-31 treatedmice versus vehicle treated mice. A similar pump delivery study was alsoperformed in normal C57BL/6 mice; mice were implanted with pumps thatdelivered 10 μg/day IL-31 or vehicle and given 2% DSS in the acuteregime. Again, there were no differences between mice in any of theDSS-colitis parameters whether implanted with IL-31 orvehicle-delivering pumps. Finally, a 2% DSS-acute colitis study wasperformed in IL-31RA deficient (IL-31RA−/−) mice. Again, there were nodifferences in body weight loss, DAI score or colon length betweenIL-31RA deficient mice and wildtype controls.

In summary, IL-31 does not appear to directly effect mucosalinflammation induced by DSS since systemic delivery of IL-31 to normalmice in acute colitis studies had no effect on disease outcome. IL-31transgenic animals may be more susceptible to DSS-induced colitis as aresult of stress caused by the transgenic phenotype. However, EμLckIL-31 transgenic mice have increased numbers of activated CD4+ and CD8+Tcells in the peripheral lymph nodes (Dillon, et al, 2004) and theincreased susceptibility to DSS-induced colitis observed in the EμLckIL-31 transgenic mice may be a consequence of the presence of theseactivated lymphocytes.

Example 8 Effects of Anti-IL31 Treatment by Sampling Dermal InterstitialFluid with Microdialysis

Microdialysis can be used with the molecules of the present invention tomeasure direct analysis of bioavailability and the distribution ofantibodies in the skin. Microdialysis is use to collect and analyze theintercellular fluid. The antibody in the interstitial fluid can bedetermined using a species-specific anti-IgG cross-linked to a luminexbead. Further, an evaluation of free to IgG-bound IL31 is done using ananti-IL31 rather than anti-IgG as the secondary antibody. 2.Proinflammatory cytokines and chemokines produced by IL31 activation ofkeratinocytes and/or dorsal root ganglion is assayed. See British J.Dermatology 142(6); 1114-1120, (2000); J. Neurol. Neurosurg. Psychiatry73; 299-302, (2002); Am J. Physiol Heart Circ. Physiol 286; 108-112,(2004); Neuroscience Letters 230; 117-120, (1997); and AAPS J. 7(3);E686-E692, (2005). See also Steinhoff, M., et al., J. Neuroscience, 23(15): 6176-6180, 2003.

Microdialysis probes are supplied by TSE Systems (Midland, Michigan).The probe is T-shaped and consists of a 3000 kDa membrane 0.3 mm OD by 4mm L attached to a 15 mm stem. The inlet and outlet are connected to0.12 mm OD peek tubing. The ex vivo analysis is performed using tubinglengths identical to that used for in vivo analysis. HMWCO probes arerun with a push/pull pump system to minimize outward (into theinterstitial) flow. However a push only (Harvard PHD 2000) is also used.Fluid loss due to Δp and ΔII is determined at various flow rates. Theefficiency (Ed) of the membrane is determined at various flow ratesusing known quantities of IgG in a mixing chamber to eliminatenon-membrane (external) diffusion. The Ed of mouse IgG and mousehemoglobin is determined and serve as in vivo controls. Quantitation isby goat anti-Rat-IgG coupled to Luminex beads and capture is reportedwith rabbit or donkey biotin-anti-rat IgG to reduce non-specificreactivity. Assays for mouse IgG and Hemoglobin is developed forcontrols in the in vivo studies. Bead coupling will be performed using astandard kit and protocol.

Treatment of mice and rats with cytokines by osmotic pump, ID or througha microdialysis fiber is used. Antibody is injected by IV. The probe isUV sterilized. The microdialysis probe is inserted and blood an analytesare sampled. Quantification of IgG transport from circulation into theskin is measured using membrane parameters determined ex vivo, antibodypermeability and the perfusion rate are estimated.

The following steps are performed using one time point per animal pairand a sufficient number of time points to estimate circulating antibodylevels and diffusion into the dermis/epidermis over time: i) amicrodialysis membrane is inserted into the skin and a preliminarysample withdrawn at a rate determined by the ex vivo analysis. Thiscontrol sample determines the baseline reactivity of the permeate fluid;2) Rat anti-IL31 antibody is introduced by IV tail injection and at thepredetermined time point an intraorbital blood sample is taken todetermine circulating antibody levels; 3) a microdialysis sample ofsufficient volume for analysis is taken at the protocol's pumping rate;4) at the end of the analyte sampling another intraorbital sample istaken to determine anti-IL31 circulating levels.

A multiplex analysis of Analyte and plasma is performed by Luminex andquantification determined for, 1.) anti-IL31 antibody, 2.)anti-mouse-IgG as a depletion/diffusion control, and 3.) anti-mouseHemoglobin to control for microdialysis insertion trauma and bloodvessel damage. Using the ex vivo determined membrane parameters and themeasured influx rate of anti-IL31 into the analyte at a givencirculating antibody concentration, an estimate of the skin diffusionrate is determined. The concentration of mouse IgG in the analyte isused to evaluate local depletion of proteins near the probe. A formulamay need to be devised to compensate for local depletion in thediffusion analysis.

Example 9 Constructs for Generating a Human IL-31Ra/OncostatinM Receptor(OSMRbeta) Heterodimer

A system for construction, expression and purification of such solubleheterodimeric receptors is known in the art, and has been adapted to thereceptor pair, human oncostatin M receptor (OSMRbeta) and human IL-31Ra.For this construct, the polynucleotide for the soluble receptor forOSMRbeta is shown in SEQ ID NO: 17 and corresponding polypeptide isshown in SEQ ID NO:18; and the polynucleotide for the soluble receptorfor human IL-31Ra is shown in SEQ ID NO:20 and corresponding polypeptideis shown in SEQ ID NO:21.

To construct a cell line expressing a secreted soluble hIL-31Ra/humanOSMRbeta heterodimer, a construct was made so that the resultingheterodimeric soluble receptor comprises the extracellular domain ofhuman OSMRbeta fused to the heavy chain of IgG gamma1 (Fc4) with aGlu-Glu tag at the C-terminus; while the extracellular domain of IL-31Rawas fused to Fc4 with a His tag at the C-terminus For both of thehIL-31Ra and human OSMRbeta arms of the heterodimer a Gly-Ser spacer of12 amino acids was engineered between the extracellular portion of thereceptor and the N-terminus of Fc4.

A. Construction of Human Soluble OSMRbeta/Fc4-CEE

For construction of the human soluble OSMRbeta/Fc4-CEE portion of theheterodimer the extracellular portion of human OSMRbeta was isolatedusing PCR with oligos under PCR reaction conditions as follows: 30cycles of 95° C. for 60 sec., 57° C. for 30 sec., and 72° C. for 100sec.; and 72° C. for 7 min. PCR products were purified using QIAquickPCR Purification Kit (Qiagen), digested with EcoR1 and BglII(Boerhinger-Mannheim), separated by gel electrophoresis and purifiedusing a QIAquick gel extraction kit (Qiagen).

The expression cassette, plasmid backbone and Fc4-GluGlu tag portion ofthe chimera were contained within a previously made in house plasmidvector. The plasmid vector was digested with EcoR1 and BamH1(Boerhinger-Mannheim), separated by gel electrophoresis and purifiedusing a QIAquick gel extraction kit (Qiagen). The digested and purifiedfragments of human OSMRbeta and Fc4-cEE containing plasmid were ligatedtogether using T4 DNA Ligase (Life Technologies, Bethesda, Md.) usingstandard ligation methods. Minipreps of the resulting ligation werescreened for an EcoRI/Sma1 insert of the correct size (772 bp) for thesoluble OSMRbeta and positive minipreps were sequenced to confirmaccuracy of the PCR reaction. This new plasmid construction is termedpZP9-ONCOMR-Fc4CEE.

B. Construction of Human Soluble IL-31Ra / Fc4-CHIS

For construction of the hIL-31Ra/Fc4-CHIS portion of the heterodimer,the extracellular portion of human IL-31Ra was isolated by digestion ofa plasmid previously containing IL-31Ra-Fc4 soluble receptor. Theplasmid was first digested with Sall (New England Biolabs, Beverly,Mass.) after which the reaction was serially phenol chloroform extractedand ethanol precipitated. The digested DNA was then treated with T4 DNAPolymerase (Boerhinger-Mannheim), to fill in the 5′ overhangs created bythe Sall digestion, leaving the DNA ends blunt, after which the reactionwas serially phenol chloroform extracted and ethanol precipitated. Theblunted DNA was then further digested with BglII to cut at the 3′ end.),separated by gel electrophoresis and purified using a QIAquick gelextraction kit (Qiagen) as per manufacturer's instruction. The resultingDNA fragment containing the sequence coding for the extracellular domainof IL-31Ra was ligated into an Fc4-CHIS tag containing mammalianexpression vector prepared as follows.

The expression cassette, plasmid backbone and Fc4-CHIS tag portion ofthe chimera were contained within a previously made in house plasmidvector. This plasmid vector was digested with EcoR1(Boerhinger-Mannheim) after which the reaction was serially phenolchloroform extracted and ethanol precipitated. The digested DNA was thentreated with T4 DNA Polymerase (Boerhinger-Mannheim), to fill in the 5′overhangs created by the EcoRl digestion, leaving the DNA ends blunt,after which the reaction was serially phenol chloroform extracted andethanol precipitated. The blunted DNA was then further digested withBamH1 (Boerhinger-Mannheim) to cut at the 3′ end, separated by gelelectrophoresis and purified using a QIAquick gel extraction kit(Qiagen). The digested and purified fragments of human IL-31Ra andFc4-CHIS containing plasmid were ligated together using T4 DNA Ligase(Life Technologies, Bethesda, Md.) using standard ligation methods.

Minipreps of the resulting ligation were screened by PCR using a IL-31Raspecific sense primer and a Fc4 specific antisense primer with thefollowing PCR reaction conditions: 30 cycles of 94° C. for 60 sec., 68°C. for 150 sec; and 72° C. for 7 min. An expected product size of 848 bpconfirmed the correct assembly of the plasmid termed pZEM228hIL-31Ra/Fc4HIS.

A second IL-31Ra-Fc4 construction was created for use in generatinghomodimer protein from COS cells. Briefly the coding region for the fullfusion protein was isolated by digestion of a plasmid previouslycontaining IL-31Ra-Fc4 soluble receptor with Sall (Boerhinger-Mannheim).The reaction was serially phenol chloroform extracted and ethanolprecipitated. The digested DNA was then treated with T4 DNA Polymerase(Boerhinger-Mannheim), to fill in the 5′ overhangs created by the EcoR1digestion, leaving the DNA ends blunt, after which the reaction wasserially phenol chloroform extracted and ethanol precipitated. Theblunted DNA was then further digested with Not1 (Boerhinger-Mannheim) tocut at the 3′ end, separated by gel electrophoresis and purified using aQIAquick gel extraction kit (Qiagen). A mammalian expression vectorcontaining a CMV driven expression cassette was digested to generatecompatible ends and the 2 fragments were ligated together. Minipreps ofthe resulting ligation were screened by PCR using vector specific senseprimer and IL-31Ra specific antisense primer with the following PCRreaction conditions: 30 cycles of 94° C. for 30 sec., 64° C. for 30 sec;70° C. for 90 sec; and 72° C. for 7 min An expected product size ofapproximately 1000 by confirmed the correct assembly of the plasmidtermed pZP7NX-hIL-31Ra-Fc4. This plasmid was subsequently transfectedinto COS cells using Lipofectamine (Gibco/BRL), as per manufacturer'sinstructions. The cells were conditioned for 60 hours in DMEM+5% FBS(Gibco/BRL) after which the protein was purified over a proteinG-sepharose 4B chromatography column and made available for in vitrobioassays, for example, such as those described herein.

C. Generating a Human IL-31Ra/OncostatinM Receptor (OSMRbeta)

About 16 μg each of the pZP9-ONCOMR-Fc4CEE and pZEM228 hIL-31Ra/Fc4HISwere co-transfected into BHK-570 (ATCC No. CRL-10314) cells usinglipofectamine (Gibco/BRL), as per manufacturer's instructions. Thetransfected cells were selected for 10 days in DMEM+5% FBS (Gibco/BRL)containing 0.5 mg/ml G418 (Gibco/BRL) and 250 nM methyltrexate(MTX)(Sigma, St. Louis, Mo.) for 10 days.

The resulting pool of doubly-selected cells was used to generate theheterodimeric protein. Three cell Factories (Nunc, Denmark) of this poolwere used to generate 10 L of serum free conditioned medium. Thisconditioned media was passed over a 1 ml protein-A column and eluted in(10) 750 microliter fractions. Four of these fractions found to have thehighest concentration were pooled and dialyzed (10 kD MW cutoff) againstPBS. The desired heterodimeric soluble IL-31Ra/OSMRbeta protein complexwas isolated from other media components by passing the pool over aNickel column and washing the column with various concentrations ofImidazole. The soluble IL-31Ra/OSMRbeta protein eluted at intermediateconcentrations of Imidazole, while hIL-31Ra/Fc4HIS homodimer eluted athigher concentrations of Imidazole.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A method of antagonizing signal transduction indorsal root ganglion cells in a subject, comprising administering to thesubject a monoclonal antibody that specifically binds to a firstreceptor consisting of amino acid residues 1-662 of SEQ ID NO:8, or asecond receptor consisting of amino acid residues 1-979 of SEQ ID NO:16,or a heterodimeric receptor comprising the first and second receptors,wherein the isotype of the monoclonal antibody is IgG, and wherein afteradministration of the monoclonal antibody, signal transduction throughthe heterodimeric receptor induced by a polypeptide comprising aminoacid residues 27-164 of SEQ ID NO:2 is reduced.
 2. The method of claim1, wherein the monoclonal antibody is humanized.
 3. The method of claim1, wherein the monoclonal antibody is human.
 4. The method of claim 1,wherein the isotype of the monoclonal antibody is IgG1, IgG2, IgG3 orIgG4.